DNA polymerase mutant having one or more mutations in the active site

ABSTRACT

This invention provides a DNA polymerase that is a mutant form of a naturally occurring DNA polymerase, of which one or more amino acids in the active site are mutated. The DNA polymerase mutant of this invention is characterized by altered fidelity or altered enzymatic activity in comparison with the naturally occurring DNA polymerase. For example, the DNA polymerase mutant provides increased enzymatic activity, altered dNTP/rNTP specificity, or enhanced fidelity. In one aspect of the invention, the naturally occurring DNA polymerase comprises an amino acid sequence motif: AspTyrSerGlnIleGluLeuArg in the active site. In another aspect of the invention, the naturally occurring DNA polymerase comprises an amino acid sequence motif: LeuLeuValAlaLeuAspTyrSerGlnIleGluLeuArg in the active site. The mutant DNA polymerase has been altered in the active site of the naturally occurring DNA polymerase to contain either (a) two or more amino acid substitutions in the amino acid sequence motif, or (b) a substitution of an amino acid other than Glu in the amino acid sequence motif.

[0001] This application is a division of U.S. application No.09/484,114, filed Jan. 14, 2000.

FIELD OF THE INVENTION

[0002] The present invention relates generally to the field of molecularbiology. Specifically, the present invention relates to a DNA polymerasethat is a mutant form of a naturally occurring DNA polymerase, in whichone or more amino acids within the active site are altered.

BACKGROUND OF THE INVENTION

[0003] DNA polymerases are responsible for the replication andmaintenance of the genome, a role that is central to accuratelytransmitting genetic information from generation to generation. DNApolymerases function in cells as the enzymes responsible for thesynthesis of DNA. They polymerize deoxyribonucleoside triphosphates inthe presence of a metal activator, such as Mg²⁺, in an order dictated bythe DNA template or polynucleotide template that is copied. Even thoughthe template dictates the order of nucleotide subunits that are linkedtogether in the newly synthesized DNA, these enzymes also function tomaintain the accuracy of this process. The contribution of DNApolymerases to the fidelity of DNA synthesis is mediated by twomechanisms. First, the geometry of the substrate binding site in DNApolymerases contributes to the selection of the complementarydeoxynucleoside triphosphates. Mutations within the substrate bindingsite on the polymerase can alter the fidelity of DNA synthesis. Second,many DNA polymerases contain a proof-reading 3′-5′ exonuclease thatpreferentially and immediately excises non-complementary deoxynucleosidetriphosphates if they are added during the course of synthesis. As aresult, these enzymes copy DNA in vitro with a fidelity varying from5×10⁻⁴(1 error per 2000 bases) to 10⁻⁷ (1 error per 10⁷ bases) (Fry andLoeb, Animal Cell DNA Polymerases), pp. 221, CRC Press, Inc., BocaRaton, Fla. (1986); Kunkel, J. Biol. Chem. 267:18251-18254(1992)).

[0004] In vivo, DNA polymerases participate in a spectrum of DNAsynthetic processes including DNA replication, DNA repair,recombination, and gene amplification (Korberg and Baker, DNAReplication, pp. 929, W. H. Freeman and Co., New York (1992)). Duringeach DNA synthetic process, the DNA template is copied once or at most afew times to produce identical replicas. In vitro DNA replication, incontrast, can be repeated many times, for example, during polymerasechain reaction (Mullis, U.S. Pat. No. 4,683,202).

[0005] In the initial studies with polymerase chain reaction (PCR), theDNA polymerase was added at the start of each round of DNA replication(U.S. Pat. No. 4,683,202). Subsequently, it was determined thatthermostable DNA polymerases could be obtained from bacteria that growat elevated temperatures, and these enzymes need to be added only once(Gelfand, U.S. Pat. No. 4,889,818). At the elevated temperatures usedduring PCR, these enzymes would not denature. As a result, one can carryout repetitive cycles of polymerase chain reactions without adding freshenzymes at the start of each synthetic addition process. DNApolymerases, particularly thermostable polymerases, are the key to alarge number of techniques in recombinant DNA studies and in medicaldiagnosis of disease. For diagnostic applications in particular, atarget nucleic acid sequence may be only a small portion of the DNA orRNA in question, so it may be difficult to detect the presence of atarget nucleic acid sequence without PCR amplification. Due to theimportance of DNA polymerases in biotechnology and medicine, it would behighly advantageous to generate DNA polymerase mutants having desiredenzymatic properties such as altered fidelity and high activity.

[0006] Polymerases contain an active site architecture that specificallyconfigures to an incorporates each of the four deoxynucleosidetriphosphates while taking direction from templates with diversenucleotide sequences. In addition, the active site tends to excludealtered nucleotides produced during cellular metabolism. The overallfolding pattern of polymerases resembles the human right hand andcontains three distinct subdomains of palm, fingers and thumb. (Beese etal., Science 260:352-355 (1993); Patel et al., Biochemistry 34:5351-5363(1995); these two references are incorporated herein by reference. Whilethe structure of the fingers and thumb subdomains vary greatly betweenpolymerases that differ in size and in cellular functions, the catalyticpalm subdomains are all superimposable. Motif A, which interacts withthe incoming dNTP and stabilizes the transition state during chemicalcatalysis, is superimposable with a mean deviation of about one Åamongst mammalian pol α and prokaryotic pol I family DNA polymerases(Wang, et al., Cell 89:1087-1099 (1997)). Motif A begins structurally atan antiparallel β-strand containing predominantly hydrophobic residuesand continues to an α-helix (FIG. 1). The primary amino acid sequence ofDNA polymerase active sites are exceptionally conserved. Motif A retainsthe sequence DYSQIELR in polymerases from organisms separated by manymillions years of evolution including Thermus aquaticus, Chlamydiatrachomatis, and Escherichia coli. Taken together, these resultsindicate polymerases function by similar catalytic mechanisms and thatthe active site of polymerases may be immutable in order to ensure thesurvival of organisms.

[0007] U.S. Pat. No. 5,939,292 is directed to a recombinant thermostableDNA polymerase that is a mutant form of a naturally occurringthermostable DNA polymerase, wherein said naturally occurringthermostable DNA polymerase has an amino acid sequence comprising aminoacid sequence motif SerGlnIleGluLeuArgXaa (SEQ ID NO:1) wherein “Xaa” atposition 7 of said sequence motif is a valine residue or an isoleucineresidue; wherein said mutant form has been modified to contain an aminoacid other than glutamic acid (Glu) at position 4 of said sequencemotif; and wherein said mutant form possesses reduced discriminationagainst incorporation of an unconventional nucleotide in comparison tosaid naturally occurring thermostable DNA polymerase. In the '292patent, the thermostable DNA polymerase mutant has an activity toincorporate ribonucleotides in vitro. The mutant has a single mutationin the active site, namely, the glutamic acid residue is altered. Webelieve that bacteria dependent on such a DNA polymerase mutant with asingle mutation of altering glutamic acid residue in the active site isnot able to survive in vivo because the mutant does not have enoughactivity for DNA replication. Our results suggest that bacteriadepending on a DNA polymerase mutant which has a Glu615 residuesubstitution will only survive if the Glu is substituted by Asp andthere is at least one additional substitution at other sites in motif A(FIG. 2). The present invention evaluates the degree of mutability of apolymerase active site in vivo. Our results counter the common paradigmthat amino acid substitutions within the catalytic site lead to reducedstability and enzymatic activity. We find that the DNA polymerase activesite is highly mutable and can accommodate many amino acid substitutionswithout affecting DNA polymerase activity significantly. The instantapplication shows that mutation on the catalytic site can produce highlyactive enzymes with altered substrate specificity. Mutant DNApolymerases may offer selective advantages such as ability to resistincorporation of chain terminating nucleotide analogs, increasedcatalytic activity, ability to copy through hairpin structures,increased processivity, and altered fidelity.

SUMMARY OF THE INVENTION

[0008] This invention is directed to a DNA polymerase that is a mutantform of a naturally occurring DNA polymerase, in which one or more aminoacids in the active site is mutated. The DNA polymerase mutant of thisinvention is characterized by altered substrate specificity, alteredfidelity or higher enzymatic activity in comparison with the naturallyoccurring DNA polymerase. A host cell dependent on the DNA polymerasemutant is able to survive and replicate repetitively. The invention alsoprovides a method of preparing a recombinant DNA polymerase that is amutant form of a naturally occurring DNA polymerase, in which one ormore amino acids in the catalytic site is mutated.

[0009] In one aspect of the invention, the naturally occurring DNApolymerase comprises an amino acid sequence motifAspTyrSerGlnIleGluLeuArg (SEQ ID NO: 2) in the active site. The mutantform has been altered to contain either (a) two or more amino acidsubstitutions in that amino acid sequence motif, or (b) one amino acidsubstitution that is not Glu in that amino acid sequence motif.

[0010] In another aspect of the invention, the naturally occurring DNApolymerase comprises an amino acid sequence motif:LeuLeuValAlaLeuAspTyrSerGlnIle GluLeuArg (SEQ ID NO: 3) in the activesite. The mutant form has been altered to contain either (a) two or moreamino acid substitutions in that amino acid sequence motif, or (b) oneamino acid substitution that is not Glu in that amino acid sequencemotif.

[0011] The present invention discovers that the active site of apolymerase is highly mutable and can accommodate many amino acidsubstitutions without affecting DNA polymerase activity. Substitutionsof amino acids within Motif A of a DNA polymerase produce enzymes withaltered catalytic activity, with altered dNTP/rNTP specificity, with lowfidelity that is capable of incorporating unconventional nucleotides,and with high fidelity that is suitable for a polymerase chain reaction.For example, the mutant DNA polymerases are characterized by the abilityto more efficiently incorporate unconventional nucleotides, particularlyribonucleotides and their analogs, than the corresponding wild-typeenzymes.

BRIEF DESCRIPTION OF THE FIGURES

[0012]FIG. 1 depicts Structure of Taq pol I bound with DNA and incomingdNTP. Evolutionarily conserved Motif A (amino acids 605 to 617highlighted in red) is located within the heart of the polymerasecatalytic site. Residues of Motif A interact with the incoming dNTP andamino acids in the finger motif during the conformational change step,subsequent to nucleotide binding. Motif A is superimposable in allpolymerases with solved structures and begins at a hydrophobic antiparallel β sheet that proceeds to an α helix. The orientation of sidechains within amino acids of Motif A is nearly identical prior (in blue)and subsequent (in red) to dNTP binding, with the exception of Asp610,which rotates around the β carbon while coordinating with the Mg⁺²-dNTPcomplex. Coordinate sets 2ktq (Taq pol I, ternary complex, open form),3ktq (ternary complex, closed form), and 4ktq (binary complex) wereobtained from Protein Data Bank.

[0013]FIG. 2 demonstrates high mutability of Motif A. The sequence ofMotif A (D⁶¹⁰YSQIELR⁶¹⁷, (SEQ ID NO: 2)) has been retained afterevolution through many millions of years in organisms such as Thermusaquaticus (SEQ ID NO: 3), Escherichia coli (SEQ ID NO: 4), and Chlamydiatrachomatis (SEQ ID NO: 5). To test the importance of this conservation,residues L605 ,to R617 were randomly mutated such that each contiguousamino acid can be replaced by potentially any of the other 19. (A) Thedegree of mutability of each amino acid within Motif A from all activeclones (>10% to 200% activity relative to wild type (WT)) complimentingan E. coli DNA polymerase I temperature sensitive strain. Amino acidsubstitutions at the locus are listed, along with the number of timeseach substitution is observed. (B) Mutations in clones exhibiting highactivity (66% to 200% WT). (C) Mutations in clones containing a singleamino acid substitution followed by activity relative to WT.

[0014]FIG. 3 compares the efficiency of dGTP and rGTP incorporation byWT and Mutant #94.

[0015]FIG. 4 shows polymerization in the presence of all 4 rNTPs with WTTaq pol I (30 fmol/μL), mutant #265 (I614N and L616I; 20 fmol/μL) andmutant #5 346 (A608D and E 615D; 20 fmol/μL). Incubation (10 μL) witheach polymerase was conducted for 10 mins at 55° C. with increasingamounts of all 4 rNTPs (0, 50, 100, 250, or 500 μM each), 23mer/46merdsDNA (primer/template; 5 nM), and 2.5 mM MgCl₂. Incubations with Mn⁺²and subsequently incubated with 0.25 N NaOH for 10 minutes at 95° C. DNAladder products resulted from incubation of thermosequenase (mutant Taqpol I) in the presence of ddNTP/dNTP mix (Amersham).

DETAILED DESCRIPTION OF THE INVENTION

[0016] The present invention provides a novel composition of a DNApolymerase that is a mutant form of a naturally occurring DNApolymerase, in which one or more amino acids in the catalytic site ismutated. The mutant DNA polymerases of this invention are active enzymeswith same or altered substrate specificity. They are characterized inaltered catalytic activity and/or altered fidelity. The low fidelitymutants are useful for introducing mutations into specific genes due tothe increased frequency of misincorporation of nucleotides during anerror-prone PCR application. The high fidelity mutants are useful forPCR amplification of genes and for mapping of genetic mutations. Themutants are therefore useful for the characterization of specific genesand for the identification and diagnosis of human genetic diseases.

[0017] To facilitate understanding of the invention, a number of termsare defined below. The term “mutant DNA polymerase” is intended to referto a DNA polymerase that contains one or more amino acids in the activesite that differ from a selected naturally occurring DNA polymerase suchas that within the Pol I family of DNA polymerases. The selected DNApolymerase is determined based on desired enzymatic properties and isused as a parent polymerase to generate a population of mutantpolymerases. For example, a thermostable polymerase such as Taq DNApolymerase I or a E. coli DNA polymerase I can be selected, for example,as a naturally occurring DNA polymerase to generate a population of DNApolymerase mutants. The “mutant DNA polymerase” of this invention is notlimited to a mutant produced by recombinant techniques; the mutant canbe produced by other methods, for example, chemical or radiationmutagenesis.

[0018] The term “catalytic activity” or “activity” when used inreference to a DNA polymerase is intended to refer to the enzymaticproperties of the polymerase. The catalytic activity includes, forexample: enzymatic properties such as the rate of synthesis of nucleicacid polymers; the K_(m) for substrates such as nucleoside triphosphatesand template strand; the fidelity of template-directed incorporation ofnucleotides, where the frequency of incorporation of non-complementarynucleotides is compared to that of complementary nucleotides;processivity, the number of nucleotides synthesized by a polymeraseprior to dissociation from the DNA template; discrimination of theribose sugar; and stability, for example, at elevated temperatures. DNApolymerases also discriminate between deoxyribonucleoside triphosphatesand dideoxyrobonucleoside triphosphates. Any of these distinct enzymaticproperties can be included in the meaning of the term catalyticactivity, including any single property, any combination of propertiesor all of the properties. The present invention includes polymerasemutants having altered catalytic activity distinct from alteredfidelity.

[0019] The term “fidelity” when used in reference to a DNA polymerase isintended to refer to the accuracy of template-directed incorporation ofcomplementary bases in a synthesized DNA strand relative to the templatestrand. Fidelity is measured based on the frequency of incorporation ofincorrect bases in the newly synthesized nucleic acid strand. Theincorporation of incorrect bases can result in point mutations,insertions or deletions. Fidelity can be calculated according to theprocedures described in Tindall and Kunkel (Biochemistry 27:6008-6013(1988)).

[0020] The term “altered fidelity” refers to the fidelity of a mutantDNA polymerase that differs from the fidelity of the selected parent DNApolymerase from which the DNA polymerase mutant is derived. The alteredfidelity can either be higher or lower than the fidelity of the selectedparent polymerase. Thus, DNA polymerase mutants with altered fidelitycan be classified as high fidelity DNA polymerases or low fidelity DNApolymerases. The term “high fidelity” is intended to mean a frequency ofaccurate base incorporation that exceeds a predetermined value.Similarly, the term “low fidelity” is intended to mean a frequency ofaccurate base incorporation that is lower than a predetermined value.The predetermined value can be, for example, a desired frequency ofaccurate base incorporation of the fidelity of a wild type DNApolymerase. Altered fidelity can be determined by assaying the parentand mutant polymerase and comparing their activities using any assaythat measures the accuracy of template directed incorporation ofcomplementary bases. Such methods for measuring fidelity include, forexample, a primer extension assay, as well as other methods known tothose skilled in the art.

[0021] The term “conventional” when referring to nucleic acid bases,nucleoside, or nucleotides refers to those which occur naturally in thepolynucleotide being described (i.e., for DNA these are DATP, dGTP, dCTPand dTTP). Additionally, c7dGTP and dITP are frequently utilized inplace of dGTP (although incorporated with lower efficiency) in in vitroDNA synthesis reactions, such as sequencing. Collectively, these may bereferred to as dNTPs.

[0022] The term “unconventional” when referring to a nucleic acid base,nucleoside, or nucleotide, includes modification, derivations, oranalogues of conventional bases, nucleosides, or nucleotides thatnaturally occur in DNA or RNA. More particularly, as used herein,unconventional nucleotides are modified at the 2′ position of the ribosesugar in comparison to conventional dNTPs. Thus, although for RNA thenaturally occurring nucleotides are ribonucleotides (i.e., ATP, GTP,CTP, UTP collectively rNTPs), because these nucleotides have a hydroxylgroup at the 2′ position of the sugar, which, by comparison is absent indNTPs, as used herein, ribonucleotides are unconventional nucleotides assubstrates for DNA polymerases. Ribonucleotide analogues containingsubstitutions at the 2′ position, such as 2′-fluoro or 2′-amino, arewithin the scope of the invention. Additionally, ribonucleotideanalogues may be modified at the 3′ position, for example, wherein thenormal hydroxyl is replaced with a hydrogen (3′ deoxy), providing aribonucleotide analogue terminator. Such nucleotides all are includedwithin the scope of the term “unconventional nucleotides. ”

[0023] Unconventional bases may be bases labeled with a reportermolecule such as a fluorophore, a hapten, a radioactive molecule or achemiluminescent molecule. For example, bases may be fluorescentlylabeled with fluorescein, or rhodamine; hapten-labeled with biotin ordigioxigenin; or isotopically labeled.

[0024] The term “expression system” refers to DNA sequences containing adesired coding sequence and control sequences in operable linkage, sothat hosts transformed with these sequences are capable of producing theencoded proteins. To effect transformation, the expression system may beincluded on a vector; however, the relevant DNA may also be integratedinto the host chromosome.

[0025] The term “gene” refers to a DNA sequence that comprises controland coding sequences necessary for the production of a recoverablebioactive polypeptide or precursor. The polypeptide can be encoded by afull-length gene sequence or by any portion of the coding sequence solong as the enzymatic activity is retained.

[0026] The term “host cell(s)” refers to both single cellular prokaryoteand eukaryote organisms such as bacteria, yeast, and actinomycetes andsingle cells from higher order plants or animals when being grown incell culture.

[0027] The mutant DNA polymerases of this invention comprises a mutationin the active site; the mutation is either a single amino acidsubstitution or multiple amino acid substitutions. The structures ofactive sites are superimposable among different naturally occurring DNApolymerases. Motif A, the active site of a DNA polymerase, whichinteracts with the incoming dNTP and stabilizes the transition stateduring chemical catalysis, is superimposable with a mean deviation ofabout one Å amongst mammalian pol I α and prokaryotic pol I family DNApolymerases. The sequence of DYSQIELR in motif A is conserved amongprocaryotic organisms such as Thermus aquaticus, Chlamydia trachomatis,and Escherichia coli. Table 1 lists the amino acid sequences of motif Aof different organisms. Of the 34 species listed, 27 comprise DYSQIELR(SEQ. ID NO: 2) in motif A, the remaining have an amino acid sequence ofDYSQIEMR (SEQ. ID NO: 6), DFSQIELR (SEQ. ID NO: 7), DYSQIELA(SEQ. ID NO:8), DYVQIELR (SEQ. ID NO: 9) or DYTQIELY (SEQ. ID NO: 10); none of thespecies have E altered in motif A. The mutant DNA polymerases of thisinvention comprises a mutation in an active site of a naturallyoccurring DNA polymerase which comprises an amino acid sequence ofDYSQIELR, DYSQIEMR, DFSQIELR, DYSQIELA, DYVQIELR or DYTQIELY. In onepreferred embodiment of the invention, the critical motif of a naturallyoccurring DNA polymerase to be modified comprises an amino acid sequenceDYSQIELR (AspTyrSerGlnIleGluLeuArg). In another preferred embodiment,the critical motif to be modified comprises an amino acid sequenceLLVALDYSQIELR (LeuLeuVal AlaLeuAspTyrSerGlnIleGluLeuArg), an amino acidsequence in motif A of Taq Pol I. The present invention also provides anisolated nucleic acid sequence encoding a DNA polymerase mutant asdescribed above. TABLE 1 Organism Motif A sequence Thermus aquaticusDYSQIELR Thermus thermophilus DYSQIELR Thermus caldophilus DYSQIELRThermus flavus DYSQIELR Thermus filiformis DYSQIELR Escherichia coli(K12) DYSQIELR Mycobacterium tuberculosis DYSQIEMR Mycobacteriumsmegmatis DYSQIEMR Mycobacterium leprae DYSQIEMR Rickettsia felisDYSQIELR Rickettsia helvetica DYSQIELR Rickettsia rhipicephali DYSQIELRRickettsia montanensis DYSQIELR Rickettsia sibirica DYSQIELR Rickettsiarickettsii (84-21C) DYSQIELR Rickettsia typhi DYSQIELR Rickettsiaprowazekii (B) DYSQIELR Rickettsia prowazekii(Madrid) DYSQIELR Bacillussubtilis DYSQIELR Bacillus stearothermophilus DYSQIELR Chlamydiatrachomatis DYSQIELR Chlamydophila pneumoniae DYSQIELR Chloroflexusaurantiacus DYSQIELR Haemophilus influenzae DYSQIELR Helicobacter pyloriDYSQIELR Lactococcus lactis DYSQIELR Methylobacterium DYSQIELRStreptococcus pneumoniae DYSQIELR Streptomyces coelicolor DYSQIELRSynechocystis sp. (PCC6803 II) DYSQIELR Aquifex aeolicus DFSQIELRBorrelia burgdorferi DYSQIELA Rhodothermus obamensis DYVQIELR Treponemapallidum DYTQIELV

[0028] By random mutagenesis protocol, a large population of mutants inwhich each amino acid is altered to potentially any of the othernineteen amino acids are created. When coupled with a stringentselection scheme, the nature of allowable amino acid substitutions invivo can be determined after sequencing selected mutants. The mutationsin motif A of an active mutant DNA polymerase include only conservativesubstitutions at sites that stabilize the tertiary structure, butinclude a wide variety of amino acid substitutions at other sites. Allthe mutants selected in this invention have at least 10% of WT DNApolymerase activity. Host cells that depend on the mutant DNApolymerases are able to live and replicate repetitively. Afterselection, plasmids containing genes that encoding active DNA polymerasemutants are purified, and nucleic acid sequences encoding the mutant DNApolymerases are determined by sequence analysis. The amino acidsequences of motif A of the mutants are derived from the nucleic acidsequences. The unique properties exhibited by the DNA polymerase mutantsinclude DNA polymerase activity higher than the wild type (WT) enzyme,the ability to incorporate unconventional nucleotides such asribonucleotides, analogs of ribonucleotides, and bases labeled withfluorescent of hapten tags. A preferred DNA polymerase mutant of thisinvention is characterized by its ability to incorporate ribonucleotidesat a rate of at least 10-fold, preferably 100-fold, and more preferably1000-fold, greater than that of WT DNA polymerase, and/or the ability tofunction as both DNA and RNA polymerases.

[0029] Sequence analysis of active mutant DNA polymerases, for example,mutants of Taq Pol I, shows that some Motif A residues tolerate a widespectrum of substitutions (Ser612, Ile614, and Arg617), some residuestolerate conservative substitutions (Tyr611, Gln613, Glu615, and Leu616), and only one residue is immutable (Asp610). Of the highly mutableresidues, Ser612, which is present in nearly all eukaryotic andprokaryotic DNA polymerases studied, tolerates substitutions that arediverse in size and hydrophilicity while often preserving WT-likeactivity. Of the other highly mutable amino acids, hydrophobic residuesLeu605 to Leu609 form a strand of the structurally conservedanti-parallel β sheet that accommodates the triphosphate portion of theincoming dNTP. Presumably those residues that tolerate conservativechanges (Tyr611, Gln613, Glu615, and Leu616) are important for dNTPbinding and/or protein stability; X-ray structure analysis show thateach of these residues has a potential role in protein interactions withimportant domains (Li, Embo, J. 17:7514-25 (1998)). Three residues(Gln613, Glu615, and Leu616) are involved in interactions with thefingers motif O helix as it changes conformation during the dNTP bindingstep and the forth (Tyr611) serves as an important anchor as well asproviding a carboxyl oxygen that binds one of the metals. The onlyimmutable residue is Asp610, which even in the context of othermutations can not be substituted even by glutamic acid. Asp610 functionsto coordinate the metal-mediated catalysis reaction, leading to theincorporation of the incoming nucleotide. The immutable nature of Asp610indicates the geometry of the active site at this precise catalyticlocus can not be altered. The analysis of mutants with a single aminoacid allows the determination of the effect on activity conferred byspecific amino acid substitutions. Leu605Arg confers greater polymeraseactivity relative to WT Taq pol I, and all selected Leu605Arg mutantsoccurring in context of multiple mutations also exhibit high activity.The single substitution, Arg617Phe, confers twice the activity of WT TaqPol I, while other substitutions at this locus lower Taq pol I activity.

[0030] A subset of mutants in our library incorporate rNTPs efficiently(Table 2). The present invention provides compositions of mutant DNApolymerases which comprise an amino acid sequence as listed in Table 2in the active site (SEQ ID NOs: 11-33). Preferred compositions of mutantDNA polymerases comprise an amino acid sequence of LLVSLDYSQNELR (SEQ IDNO: 14), LLVALDYSQNEIR (SEQ ID NO: 21) or LLVDLDYSQIDLR (SEQ ID NO: 24)in the active site. The mutants of Table 2 contain 1, 2, 3 and 4 aminoacid substitutions and fall into two major classes: 1) Those encoding ahydrophilic substitution at Ile614; these enzymes constitute themajority of rNTP incorporating mutants with 1 or 2 substitutions, and 2)those that encode a Glu615Asp substitution; these enzymes contain 1-3other substitutions and have a total of 2-4 substitutions. None of ourmutants contain a single Glu615 substitution. Our results suggest thatGlu615 is important for dNTP binding and DNA polymerase activity. Asingle mutation in motif A which alters the glutamic acid may fatallyimpair the DNA polymerase activity. A conservative substitution of Gluto Asp plus additional compensating mutations in motif A may provide aproper tertiary structure for the DNA polymerase activity. Kineticanalysis shows purified WT Taq pol I does not efficiently incorporateribonucleotides. Our DNA polymerase mutants incorporate eachribonucleotide up to three orders of magnitude more efficiently than theWT polymerase. TABLE 2 Sequences of rNTP incorporating Taq polymerases614 615 617 WT L L V A L D Y S Q I E L R #aa Δs Mutant 1  53† K  75 M 2 65 D P  94 S N 164 V K 187 L K 198 M Q 205 L Q 221 V D 230 R F 265 N I273 G D 340 V D 346 D D 3  79 D V D 159 I L K 166 M M D  175‡ F T D 298V V K 299 T F W  300‡ F T D 4  6 M V V D  48 R K D M

[0031] We propose two mechanisms by which the steric interferenceconferred by Glu615 on an incoming ribonucleotide (FIG. 1) can bealleviated while still allowing utilization of dNTPs. 1) Hydrophilicsubstitutions at Ile614 could alter the steric environment byinteracting with and repositioning the adjacent Glu615. 2) The Glu615Aspsubstitution reduces the length of the side chain and diminishesblockage while still allowing the essential hydrogen bonding to thehelix O residue Tyr671.

[0032] To determine if the polymerases mutant can function as RNApolymerases by incorporating multiple ribonucleotides sequentially, thepurified WT Taq pol I, a mutant containing substitution at I614, and amutant containing a substitution at E615, are incubated with increasingamounts of all four rNTPs. While the WT enzyme inefficientlyincorporates and extends ribonucleotides, both classes of rNTP utilizingmutant enzymes polymerize multiple ribonucleotides, even at rNTPconcentrations well below that found in cells. In control incubationsthe elongated products can be degraded in alkali to regenerate theinitial substrate, illustrating the products are RNA. Thus, our randommutagenesis protocol has identified a set of DNA polymerases containing1-2 gain of function mutations conferring the ability to incorporatesuccessive ribonucleotides. Even though these mutants may confer areduced fitness to the cells over long term by incorporatingribonucleotides into chromosomal DNA, the observation that 23independent rNTP incorporating mutants are selected using a DNApolymerase-deficient strain indicates that a functioning DNA polymeraseis important for survival, even if this polymerase transientlyincorporates ribonucleotides during the first >50 generations.

[0033] The present invention provides mutant DNA polymerases suitablefor use with ribonucleoside triphosphates for numerous applicationsincluding nucleic acid amplification, nucleic acid detection and DNAsequencing analysis. The use of ribonucleotides in sequencing avoids thehigh cost of chain-terminating analogues, such as ddNTPs. In addition,it facilities the preparation of novel amplification products suitablenot only for DNA sequence analysis but also for other types of analysissuch as electrophoresis or hybridization without the need to conductsubsequent DNA sequencing reactions.

[0034] The present invention provides a mutant DNA polymerase that canincorporate a reporter-labeled nucleotide analog, for use in diagnosisof disease. In this application, DNAs from specific pathogens such asbacteria or viruses can be detected from a clinical sample (e.g., blood,urine, sputum, stool, sweat, etc.) The sample is first heated to exposeits genome and to denature its DNAs. Next, a small single-stranded DNAfragment that is complementary to a region of the pathogen's genome isadded such that the DNA fragment can hybridize with a complementaryregion of the pathogen's genomic DNAs. Then, a mutant DNA polymerase ofthe present invention that can efficiently incorporate areporter-labeled nucleotide analog is added in the presence of all fourdNTPS and a trace amount of a reporter-labeled nucleotide analog. Thereporter molecule can be a fluorophore such as fluorescein, Texas red,rhodamine, Cascade Blue dye, etc., a hapten such as biotin ordigioxigenin, a radiolabel, or a chemiluminescent molecule. Extension ofthe small-hybridized DNA fragment by the mutant DNA polymnerase resultsin a “tagged” DNA fragment. The presence of an abundant amount of taggedDNAs signifies the presence of a specific pathogen. This protocol can bemodified by fluorescently labeling many different sets of smallsingle-stranded DNA; each contains a different fluorophore and exhibitsa different emission spectrum (e.g., red, blue, magenta, yellow, etc.)Each small single-stranded DNA can hybridize to the genome of one of themany distinct pathogenic agents. Following DNA synthesis by a mutantpolymerase in the presence of a uniquely fluorescently labelednucleotide, a specific pathogen can be diagnosed by determining thenature of the fluorescent signal from the extended DNAs.

[0035] The present invention provides a mutant DNA polymerase that has ahigher fidelity comparing with a WT DNA polymerase. The mutant DNApolymerase are useful in copying or repetitive DNA sequences, for theapplication in cancer diagnostics, and in gene therapy/cancer therapy tokill tumors via incorporation of toxic analogs.

[0036] The present invention also provides mutant DNA polymerases havingenhanced fidelity compared with WT DNA polymerase. For example, onemutant with six substitutions (Leu610Arg, Leu606Met, Val607Lys,Ala608Ser, Leu609Ile and Ser612Arg) exhibits about 5-fold higherfidelity than the WT Taq Pol I. The invention provides a method of usinghigh fidelity DNA polymerase mutants, which comprise a mutation in theactive site, for amplifying a specific nucleic acid sequence in apolymerase chain reaction,. The polymerase chain reaction is describedin detail in U.S. Pat. No. 4,683,202; the reference is incorporatedherein by reference. Briefly, the specific nucleic acid sequenceconsists of two separate complementary strands and is contained in anucleic acid or a mixture of nucleic acids. The amplification methodcomprises the steps of: (a) treating the two strands with twooligonucleotide primers in the presence of a high fidelity mutant DNApolymerase, under conditions such that an extension product of eachprimer is synthesized which is complementary to each nucleic acid strandof the specific nucleic acid sequence, wherein said primers are selectedso as to be sufficiently complementary to the two strands of thespecific sequence to hybridize therewith, such that the extensionproduct synthesized from one primer, when it is separated from itscomplement, can serve as a template for synthesis of the extensionproduct of the other primer; (b) separating the primer extensionproducts from the templates on which they were synthesized to producesingle-stranded molecules; and (c) treating the single-strandedmolecules generated from step (b) with the primers of step (a) in thepresence of the mutant DNA polymerase, under conditions that a primerextension product is synthesized using each of the single strandsproduced in step (b) as a template. In a preferred method, step (b) isaccomplished by denaturing such as heating. One of the DNA polymerasemutants suitable for the PCR application comprises an amino acidsequence of RMKSIDYRQIELR (SEQ ID NO: 34).

[0037] A mutant DNA polymerase of the present invention have a molecularweight in the range of 85,000 to 105,000, more preferably between 90,000to 95,000. The amino acid sequence of these polymerases consists ofabout 750 to 950 amino acid residues, preferable between 800 and 900amino acid residues. The polymerases of the present invention may alsoconsist of about 540 or more amino acids and comprise at least thepolymerase domain, and a portion corresponding to the 3′ to 5′exonuclease domain and possibly parts of the 5′ to 3′ exonucleasedomain, which is contained on the first one-third of the amino acidsequence of many full-length thermostable polymerase enzymes.

[0038] Exemplary mutant DNA polymerases of the present invention arerecombinant derivatives of the native polymerases from the organismslisted in Table 1. Table 1 also indicates the particular sequence of thecritical motif in which a mutation occurs. For DNA polymerases not shownin Table 1, preparing a mutant polymerase is simple once the criticalmotif in the amino acid sequence is identified.

[0039] The invention provides a method for identifying a mutant DNApolymerase having altered fidelity or catalyic activity. The methodconsists of generating a random population of polymerase mutants bymutating at least one amino acid residue in motif A of a naturallyoccurring DNA polymerase and screening the population for activepolymerase mutants by genetic selection.

[0040] The generation and identification of polymerases having alteredfidelity or altered catalytic activity is accomplished by first creatinga population of mutant polymerases comprising randomizedoligonucleotides within motif A. The identification of active mutants isperformed in vivo and is based on genetic complementation of conditionalpolymerase mutants under non-permissive conditions. Once identified, theactive polymerases are then screened for fidelity of polynucleotidesynthesis and for catalytic activity.

[0041] The methods of the invention employ a population of polymerasemutants and the screening of the polymerase mutant population toidentify an active polymerase mutant. Using a population of polymerasemutants is advantageous in that a number of amino acid substitutionsincluding a single amino acid substitution and multiple amino acidsubstitutions can be examined for their effect on polymerase fidelity.The use of a population of polymerase mutants increases the probabilityof identifying a polymerase mutant having a desired fidelity.

[0042] Screening a population of polymerase mutants has the additionaladvantage of alleviating the need to make predictions about the effectof specific amino acid substitutions on the activity of the polymerase.The substitution of single amino acids has limited predictability as toits effect on enzymatic activity and the effect of multiple amino acidsubstitutions is virtually unpredictable. The methods of the inventionallow for screening a large number of polymerase mutants which caninclude single amino acid substitutions and multiple amino acidsubstitutions. In addition, using screening methods that select foractive polymerase mutants has the additional advantage of eliminatinginactive mutants that could complicate screening procedures that requirepurification of polymerase mutants to determine activity.

[0043] Moreover, the methods of the invention allow for targeting ofamino acid residues adjacent to immutable or nearly immutable amino acidresidues. Immutable or nearly immutable amino acid residues are residuesrequired for activity, and those immutable residues located in theactive site provide critical residues adjacent to these requiredresidues provides the greatest likelihood of modulating the activity ofthe polymerase. Introducing random mutations at these sites increasesthe probability of identifying a mutant polymerase having a desiredalteration in activity such as altered fidelity.

[0044] A naturally occurring DNA polymerase is selected as a parentpolymerase to introduce mutations for generating a library of mutants.Polymerases obtained from thermophlic organisms such as Thermusaquaticus have particularly desirable enzymatic characteristics due totheir stability and activity at high temperatures. Thermostablepolymerases are stable and retain activity at temperatures greater thanabout 37° C., generally greater than about 50° C., and particularlygreater than about 90° C.. The use of the thermostable polymerase TaqDNA polymerase I as a parent polymerase to generate polymerase mutantsis disclosed herein in the Examples.

[0045] In addition to creating mutant DNA polymerases from organismsthat grow at elevated temperatures, the methods of the invention cansimilarly be applied to non-thermostable polymerases provided that thereis a selection or screen such as the genetic complementation of aconditional polymerase mutation. Such a selection or screen of anon-thermostable polymerase can be, for example, the inducible ofrepressible expression of an endogenous polymerase. Polymerases havingaltered fidelity or altered catalytic activity can similarly begenerated and selected from both prokaryotic and eukaryotic cells aswell as viruses. Those skilled in the art will know how to apply theteachings described herein to the generation of polymerases havingaltered fidelity from such other organisms and such other cell types.

[0046] Although a specific embodiment using Taq DNA polymerase I isdisclosed in the examples, the methods of the invention can similarly beapplied to DNA polymerases other than Thermus aquaticus DNA polymerases.Such other polymerases include, for example, Escherichia coli,Mycobacterium, Rickettsia, Bacillus, Chlamydia, Chlamydophila,Chloroflexus, Haemophilus, Helicobacter, Lacococcus, Methylobacterium,Streptococcus, Streptomyces, Synechocysts, Aquifex, Borielia,Rhodothermus, and Treponema. Using the guidance provided herein inreference to Taq DNA polymerases, those skilled in the art can apply theteachings of the invention to the generation and identification of theseother polymerases having altered fidelity of polynucleotide synthesis.

[0047] Thus, the invention provides a general method for the productionof a DNA polymerase mutant that has an altered fidelity or an alteredcatalytic activity in DNA synthesis. The altered polymerase fidelity canbe either an increase or a decrease in the accuracy of DNA synthesis. Anexample of a preferred DNA polymerase mutant has an altered substratespecificity.

[0048] In one embodiment, the invention involves the production of apopulation nucleic acids encoding a polymerase with altered motif A andintroduction of the population into host cells to produce a library. Themutagenized polymerase encoding nucleic acids are expressed, and thelibrary is screened for active polymerase mutants by complementation ofa temperature sensitive mutation of an endogenous polymerase. Colonieswhich are viable at the non-permissive temperature are those which havepolymerase encoding nucleic acids which code for active mutants.

[0049] The modified gene or gene fragment can be recovered from theplasmid, or phage by conventional means and ligated into an expressionvector for subsequent culture and purification of the resulting enzyme.Numerous cloning and expression vectors, including mammalian andbacterial systems, are suitable for practicing the invention, and aredescribed in, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, second edition, Cold Spring Harbor, 1989. Those ofskill in the art will recognize that the mutant DNA polymerases withdifferent activities from the wild type enzyme are most easilyconstructed by recombinant DNA techniques. When one desires to produceone of the mutant enzymes of the present invention, or a derivative orhomologue of those enzymes, the production of a recombinant form of theenzyme typically involves the construction of an expression vector, thetransformation of a host cell with the vector, and culture of thetransformed host cell under conditions such that expression will occur.Means for preparing expression vectors, transforming and culturingtransformed host cells are well known in the art and are described indetail in, for example, Sambrook et al., 1989, supra.

[0050] To generate a random population of polymerase mutants, a randomsequence of nucleotides is substituted for motif A sequence of aplasmid-encoded gene that specifies a DNA polymerase. In one applicationof this procedure, a partial double-stranded DNA is created with 3′recessed-ends by hybridizing a first oligodeoxyribonucleotide containinga defined sequence with a restriction site “X”. This firstoligodeoxyribonucleotide is hybridized to a secondoligodeoxyribonucleotide, which contains a nucleotide sequencecomplementary to the defined sequence and a partially randomizedsequence encoding amino acids of interest. The secondoligodeoxyribonucleotide additionally contains a restriction site “Y”.The partially double-stranded oligonucleotide is filled in by DNApolymerase, cut at restriction sites “X” and “Y”, and ligated into avector. After ligation, the reconstructed plasmids constitute a libraryof different nucleic acid sequences encoding the thermostable DNApolymerase and polymerase mutants.

[0051] A genetic screen can be used to identify active polymerasemutants. For example, the library of nucleic acid sequences encoding TaqDNA polymerase and polymerase mutants are transfected into a bacterialstrain such as E. coli strain recA718polA12, which contains atemperature sensitive mutation in DNA polymerase. Exogenous DNApolymerases have been shown to functionally substitute for E. coli DNApolymerase I using E. coli strain recA718polA12 and to complement theobserved growth defect at elevated temperature, presumably caused by theinstability of the endogenous DNA polymerase I at elevated temperatures(Sweasy and Loeb, J Biol. Chem. 267:1407-1410 (1992); Kim and Loeb,Proc. Natl. Acad. Sci USA 92:684-488 (1995)). Using a complementationsystem, which employs a randomly mutated Taq library to complement thegrowth defect of E. coli strain recA718polA12, Taq DNA polymerase Imutants are identified in host bacteria that harbor plasmids encodingactive thermoresistant DNA polymerases that allow bacterial growth andcolony formation at elevated or restrictive temperatures.

[0052] In addition, active and thermostable mutants can be identified bylysing thermolabile bacteria host (e.g. E. coli) and analyzing directlyfor DNA polymerase activity at elevated temperatures. For example,active Taq polymerase mutants can be screened for the ability tosynthesize DNA (e.g., by incorporating radioactive nucleotides) at anelevated temperature. This method can be expanded for screening otheractive thermostable enzyme mutants expressed in thermolabile hosts. Inthe method, individual mutants from a random library are expressed inthermolabile hosts. Colonies of E. coli harboring a unique mutantprotein of interest are propagated at 37° C. The mutant protein ispartially purified by heat denaturing and lysing the host bacteria atelevated temperatures such as 95° C. Following centrifugation, thesupernatant containing partially purified thermostable protein ofinterest can be collected and tested for a specific activity of theprotein. In our studies with various enzymes, we have identified that5-10% of random mutants containing substitutions within the catalyticsite are active. Thus, this screen method is potentially useful for manythermostable protein.

[0053] The production of mutant DNA polymerases with active enzymaticactivities may also be accomplished by processes such as site-directedmutagenesis. See, for example, Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor, 1989, second edition, Chapter15.51, “Oligonucleotide-Mediated Mutagenesis,” which is incorporatedherein by reference. Site-directed mutagenesis is generally accomplishedby site-specific primer-directed mutagenesis. This technique is nowstandard in the art and is conducted using a synthetic oligonucleotideprimer complementary to a single-stranded phage DNA to be mutagenizedexcept for a limited mismatch representing the desired mutation.Briefly, the synthetic oligonucleotide is used as a primer to directsynthesis of a strand complementary to the plasmid or phage, and theresulting double-stranded DNA is transformed into a phage-supportinghost bacterium. The resulting bacteria can be assayed by, for example,DNA sequence analysis or probe hybridization to identify those plaquescarrying the desired mutated gene sequence. Alternatively, “recombinantPCR” methods can be employed (Innis et al. editors, PCR Protocols, SanDiego, Academic Press, 1990, Chapter 22, Entitled “Recombinant PCR”,Higuchi, pages 177-183).

[0054] The fidelity of active polymerase mutants can be determined byseveral methods. The active polymerases can be, for example, screenedfor altered fidelity from crude extracts of bacterial cells grown fromthe viable colonies. In one method, a primer extension assay is usedwith a biased ratio of nucleoside triphosphates consisting of only threeof the nucleoside triphosphates. Elongation of the primer past templatepositions that are complementary to the deleted nucleoside triphosphatesubstrate in the reaction mixture results from errors in DNA synthesis.Processivity of high fidelity polymerases will terminate when theyencounter a template nucleotide complementary to the missing nucleosidetriphosphate whereas the low fidelity polymerases will be more likely tomisincorporate a non-complementary nucleotide. The accuracy ofincorporation for the primer extension assay can be measured by physicalcriteria such as by determining the size or the sequence of theextension product. This method is particularly suitable for screeningfor low fidelity mutants since increases in chain elongation are easilyand rapidly quantitated.

[0055] A second method for determining the fidelity of polymerasemutants employs a forward mutation assay. A template containing a singlestranded gap in a reporter gene such as lacZ is used for the forwardmutation assay. Filling in of the gapped segment is carried out by crudeheat denatured bacterial extracts harboring plasmids expressing athermostable DNA polymerase mutant. For determining low fidelitypolymerase mutants, reactions are carried out in the presence ofequimolar concentrations of each nucleoside triphosphate. Fordetermining high fidelity polymerase mutants, the reaction is carriedout with a biased pool of nucleoside triphosphates. Using a biased poolof nucleoside-triphosphates results in incorporation of errors in thesynthesized strand that are proportional to the ratio ofnon-complementary to complementary nucleoside triphosphates in thereaction. Therefore, the bias exaggerates the errors produced by thepolymerases and facilitates the identification of high fidelity mutants.The fidelity of DNA synthesis is determined from the number of mutationsproduced in the reporter gene.

[0056] Procedures other than those described above for identifying andcharacterizing the fidelity of a polymerase are known in the art and canbe substituted for identifying high or low fidelity mutants. Thoseskilled in the art can determine which procedures are appropriatedepending on the needs of a particular application.

[0057] Our results counter the common paradigm that amino acidsubstitutions within the catalytic site lead to reduced stability andenzymatic activity. Our genetic selection protocol allows isolation ofmutant polymerases that retain a high DNA polymerase activity. Bacteriadependent on these polymerases can be grown under logarithmic conditionsin liquid broth (prior to plasmid isolation and protein purification) oras colonies in solid agar at 37° C. (>50 generations) withoutsignificant variations in growth kinetics. Thus bacteria dependent onmutant enzymes for survival are fit to replicate repetitively. MutantDNA polymerases may offer selective advantages such as: ability toresist incorporation of chain terminating nucleotide analogs, increasedcatalytic activity, ability to copy through hairpin structures,increased processivity, and altered fidelity. For example, some mutantsin our library are more active than WT Taq pol I, and some mutantsexhibit enhanced fidelity. Some mutants can incorporate chemotherapydrugs such as ara-C and acyclovir 100 times more efficiently than wt Taqpol I.

[0058] We find, following random sequence mutagenesis and selection bygenetic complementation, that amino acids of the polymerase active siteare highly mutable. Our studies produced highly active enzymes.Preservation of a plastic, mutable active site could facilitate thegeneration of beneficial mutants under specific selective forces such asmutant polymerases able to transiently incorporate ribonucleotides ortheir analogs under conditions of dNTP deprivation duringnucleotide-based therapy. Such ribonucleotide analogs include ara-C,acyclovir, or other antiviral or anti-cancer drug. In addition, theplastic nature of active sites may allow proteins to tolerate highmutation burdens. It has been demonstrated that as few as threesuccessive selection steps yielded a population of E. coli cells thatmutated at elevated rates (Mao, et al., J Bacteriol. 179:417-422(1997)), and 1-5% of pathogenic E. coli and Salmonella enterica aremutators (LeClerc, et aL, Science 274:1208-11 (1996)). Enrichment formutator cells under adverse conditions could account for the generationof a mutator phenotype during cancer progression (Loeb, Science277:1449-150 (1997)). In addition, exponential growth ofrecombination-incompetent E. coli after four years yields populationswith heterogeneous genotypes (Elena, et al., Science 272:1802-4 (1996)).

[0059] GenBank sequence alignment analysis of over 20 polA genes fromdifferent organisms show that a large majority of the organisms haveretained the DYSQIELR motif within the pol I active site, and specieswithin a genus have retained up to 90% sequence identity for the entirepolymerase gene. Thus, DNA polymerase sequence appears to be homogeneousafter millions of generations. Amino acid sequence identity can bepreserved by one of at least two mechanism. 1) WT amino acid sequencemay have the highest over all fitness and thus selective advantage overmutated sequences, or 2) recombination-like mechanisms serve to preservehomogeneous sequences. The predominance of one mechanism over the othercan be differentiated by examining the nucleotide sequence in additionto the amino acid sequence. Selection of WT amino acid sequence can leadto accumulation of silent mutations after prolonged evolution thatencode for identical amino acids. In contract, if horizontal transfer ofgenetic material serves to preserve homogeneous amino acid sequences,then the nucleotide sequences should also be homogeneous. Sequencealignments of E. coli polA gene encoding DNA polymerase I from distinctstrains (K-12 and B) dividing independently for many years and relatedspecies within the same genus (e.g. Thermus acquaticus and Thermusthermophilus; Mycobacterium tuberculosis and Mycobacterium smegmatis;Rickettsia) which have been evolving separately for many years show eachmember has nearly identical nucleotide sequence. Thus, related organismshave maintained relatively homogeneous genomes after many milliondivisions. From this information, a more detailed model of punctuatedevolution would allow for: 1) Growth during adverse conditions selectsfor populations of mutators; 2) Inherent plasticity of proteins wedescribe here enables tolerance of the high mutation burden duringadverse conditions and the generation of mutations with a selectiveadvantage; 3) Following successful survival through periods of adverseconditions, WT sequence (one that is fit and the most prevalent) isgenerated through horizontal transfer.

[0060] The following examples are offered by way of illustration onlyand are by no means intended to limited the scope of the claimedinvention.

EXAMPLES

[0061] Example 1. Preparing Plasmids Containing Substituted Random DNASequences from Leu605 to Arg617 of Thermus aguaticus DNA Polymerase I.

[0062] Taq pol was cloned into low copy (1 to 3 copies/cell) pHSG576vector containing a E. coli pol I independent origin of replication,SC101. A silent BisWI site was created in Taq pol I by site directedmutagenesis (C to A) at position 1758 (pTaq). A nonfunctional stuffervector (pTaqDUM) was constructed by cloning two hybridized oligos intopTaq between BisWI and SacII sites; these two restriction sites flankthe sequence encoding for Motif A. A random library (pTaqLIB) wascreated by preparing a randomized oligo with a BisWI site in whichnucleotides encoding amino acids Leu605 to Arg 617 contained 88%wild-type and 4% each of the other three nucleotides. This oligo washybridized with an oligonucleotide primer containing SacII site inequimolar proportions, and T7 DNA polymerase (exo-) was used to copy thetemplate containing the randomized nucleotides. The double-strandedoligo was digested with BisWI and SacII, purified, and inserted intopTaqDUM between BisWI and SacII restriction sites in place of thestuffer fragment. The reconstructed plasmids were transformed into DH5acells by electroporation, and the cells were incubated in 1 mL 2×YT(yeast Tryptone media) at 37° C. for 1 hour. The number of clones withinthe library was determined by plating an aliquot onto 2×YT platescontaining 30 μg/mL chloramphenicol. The remainder of the transformationmixture was pooled and incubated in 1 L of 2×YT containingchloramphenicol for 12 hours at 37° C. Plasmids were purified (pTaqLIB)by CsC1 gradient centrifugation.

[0063] Example 2. Selecting Active Clones by Genetic Complementation.

[0064] In complementation studies, E. coli recA718polA12 cells wereused. This E. coli strain, which contains a temperature sensitivemutation in polA gene encoding DNA polymerase I, forms colonies at 30°C., but not at 37° C. The E. coli recA718polA12 cells were transformedwith 0.2μg each of the following plasmids: pHSG576, pTaqDUM, pTaq, orpTaqLIB by electroporation, and the cells were allowed to recover innutrient broth medium for 2 hours at 30° C. Following recovery, a smallfraction of the mixture was plated in duplicate onto nutrient agarplates containing chloramphenicol; one plate was incubated at 30° C. andthe other at 37° C. for 24 hrs, and resulting colonies were counted.Only paired samples that contained 200 colonies or less at 30° C. wereanalyzed, because dense plating of cells leads to elevated background at37° C. Complementation experiments with either inactive pHSG576 orpTaqDUM consistently yielded over 100-fold fewer colonies at 37° .Crelative to 30° C., indicating that the background for ourcomplementation-based section assay <1%. Transformation with pTaqconsistently yields equal number of colonies after incubations at 30 or37° C., indicating that Taq pol I fully restores the growth defectivephenotype at the elevated temperatures, of 37° C.

[0065] We constructed a randomly mutated Taq library containing 200,000independent clones, and 5% of the transformed E. coli recA718polA12formed colonies at 37 ° C. relative to 30 ° C. After subtracting thebackground (<1%), we estimate there are 8,000 to 10,000 independentlibrary clones that encode an active Taq pol I. This alone suggests thatthe polymerase catalytic site can potentially accommodate a surprisinglylarge number of amino acid substitutions in vivo.

[0066] Example 3. Sequencing the Randomized Insert from UnselectedClones.

[0067] To establish the spectrum of mutations that restored growth of E.coli recA718 polA12, we sequenced the randomized insert from bothunselected clones (30 ° C.) and from selected clones (37 ° C.). Plasmidsharboring WT and mutant Taq pol Is were isolated by minipreps (Promega)after overnight propagation at 37° C. in 2×YT, and 200 nts surroundingthe randomized region were amplified by PCR and sequenced. Analysis ofsequences from unselected plasmids, which reflects the distribution ofmutants found in the random library prior to selection, shows that theaverage number of amino acid (amino acid) substitution is 4.

[0068] Of the 26 unselected clones we sequenced, 3 clones have 2 aminoacid substitutions; 4 clones have 3 aa changes, 7 have 4 aa changes, 4have 5 aa changes, 1 has 7 aa changes; 1 contains an insertion, 4contain deletions, and 2 are pTacDUM.

[0069] Example 4. Sequencing and Measuring Activities from SelectedClones.

[0070] After selection, we randomly picked 350 colonies that grew at 37° C., measured the Taq DNA polymerase activity, isolated the plasmidsand sequenced 200 nucleotides encompassing the substituted randomsequence.

[0071] The 350 colonies that grew on 37° C. plates were isolated andgrown in nutrient broth individually overnight at 30° C. Each culturewas grown to O.D. of 0.3 at 30° C. in 10 mL and Taq pol I expression wasinduced with 0.5 mM IPTG and incubations continued for 4 hours. Taq polswere partially purified using a modified protocol of refs. (Grimm, etal., Nucleic Acids Res 23:4518-9 (1995), Desai, et al., Biotechniques19:780-2,784 (1995)), which allows efficient (>50% purification of Taqpol I while removing endogenous polymerase and nuclease activities.Polymerase activity was assayed using a 20 μL reaction mixturecontaining 50 mM KC1, 10 mM Tris-HC (pH 8), 0.1% Triton-X, 2.5 mM MgCl₂,0.4 mg activated calf thymus DNA, 10 μM each dNTP, 0.25 mCi [α-³²P]dATP,and 1 μL of partially purified WT or mutant Taq pols. Incubations wereat 72° C. for 5 min and reactions were stopped with the addition of 100μL 0.1 M sodium pyrophosphate, followed by 0.5 mL 10% TCA. Polymeraseactivity was quantified by collecting precipitated radioactive DNA ontoglass filter papers, and amount of radioactive counts were measured byscintillation.

[0072] Of the 350 clones, 20 were inactive (<2% DNA polymerase activityrelative to WT); 39 clones had low activity (2 to 10%) and/orthermostability; while 291 were active (>10 to 200% WT activity). The291 independent active clones had on average 2 amino acid changes,ranging from no amino acid changes (27 clones) to one clone containing 6amino acid changes; two clones have ambiguous sequences. Taq pol I fromthe 27 plasmids that encode WT enzyme at the same amino acid sequence(yet containing silent nucleotide changes) have similar DNA polymeraseactivity relative to WT controls. The preparations from pTaqDum and pHSGnegative controls yield <1% of WT polymerase activity. Of the 60 mutantswith a single amino acid change, 60% (36 mutants) are highly active(>66% to 200% WT activity). In comparison, 27% (24 out of 90 mutants)with 2 amino acid changes, 20% (14 out of 70 mutants) with 3 amino acidchanges, 22% (7 out of 32) with 4 amino acid changes, 11% (1 out of 9)with 5 amino acid changes and a single mutant with 6 amino acid changes,were all highly active. Thus, even in cases of especially pronouncedmutation burden with one-third to one-half of an evolutionarilyconserved motif altered, a large number of mutants exhibit highactivity. These 263 clones containing 1 to 6 amino acid substitutionsrepresent a large collection of physiologically active polymerasemutants.

[0073] Sequence analysis of all 291 selected active clones (10 to 200%WT activity, FIG. 2A), including 87 most active mutants (>66% to 200% WTactivity, FIG. 2B), showed that most Motif A residues tolerated a widespectrum of substitutions (Leu605, Leu606, Val607, Ala608, Leu609,Ser612, Ile614, and Arg617), some residues tolerated conservativesubstitutions (Tyr611, Gln613, Glu615, and Leu 616), and only oneresidue was immutable (Asp610). One of the highly mutable residues,Ser612, tolerated substitutions that were diverse in size andhydrophilicity while often preserving WT-like activity. A mutant with 6substitutions (Leu605Arg, Leu606Met, Val607Lys, Ala608Ser, Leu609Ile,and Ser612Arg) exhibited WT DNA polymerase activity. Analysis of 59mutants with a single amino acid change (FIG. 2C) yielded a similardistribution of mutability and allowed us to determine the effect onactivity conferred by specific amino acid substitutions. Leu605Argconferred greater polymerase activity relative to WT Taq pol I (150%),and all selected Leu605Arg mutants occurring in context of multiplemutations also exhibited high activity (FIGS. 2A and 2B). The singlesubstitution, Arg617Phe, confers twice the activity of WT Taq Pol I,while other substitutions at this locus lowered Taq pol I activity (FIG.2C).

[0074] Example 5. Screening Selected Clones for the Ability toIncorporate Ribonucleotides.

[0075] To determine if alterations within the catalytic site can conferother properties on a DNA polymerase and lead to alterations in thesubstrate specificity, we screened all 291 selected clones for theability to incorporate ribonucleotides.

[0076] Each of the selected Taq pols that retain at least 10% activityrelative to WT enzyme at 72 ° C. (291 total) were tested for the abilityto incorporated ribonucleotides. Primer/template constructs wereprepared by hybridizing 5′- ³²P end-labeled 23 mer primer (5′cgc gcc gaattc ccg cta gca at, SEQ ID NO: 35) with 46 mer template (5′-gcg cgg aagctt ggc tgc aga ata ttg cta gcg gga att cgg cgc g, SEQ ID NO: 36) usinga 1:2 primer to template ratio. The primer/template (5 nM) was incubatedin the presence of 50 mM KC1, 10 mM Tris-HC1 (pH 8), 0.1% Triton-X, 2.5mM MgC1₂, and 1 μL of partially purified Taq pols (0.1 to 0.01 units) in10 μL volumes in the presence of 0 to 250 μM each rNTPs. Reactions wereterminated after 30 min incubation at 55 ° C. with the addition of 2 μLof formamide containing stop solution (Amersham). Products were analyzedby 14% denaturing PAGE.

[0077] This screen identified a small subset of mutants (23 out of 291)that can incorporate rNTPs efficiently (Table 2). These 23 mutants fallinto two major classes: 1) Those encoding a hydrophilic substitution atIle614; these enzymes constitute the majority of rNTP incorporatingmutants with 1 or 2 substitutions, and 2) those that encode a Glu615Aspsubstitution; these enzymes contain 1-3 other substitutions.

[0078] Example 6. Purifying Wild Yype and Mutant Tag Polymerase.

[0079] Wild type and mutant (#94, #265, and #346; Table 2) Taq pols werepurified to homogeneity using a modified procedure according to Engelke,et al., (Anal Biochem 191: 396-400 (1990)). Step 1: Bacteria cultures(DH5α cells; 2L) harboring pTaq or selected mutant pTaqLIB plasmid wereharvested and lysed in the presence of buffer A (30 mM Tris-HC1, pH 7.9,50 mM glucose, 1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.5%Tween 20, 0.5% Nonident P40) with lysozyme (4 mg/mL) with repetitivefreezing and thawing at −70 ° C. and 70 ° C. Step 2: Taq pol wasprecipitated by the addition of polyethyeneimine at a finalconcentration of 0.1%; recovered by centrifugation and washed withbuffer containing low salt (0.025 M KC1) buffer C (20 mM HEPES, pH 7.9,1 mM EDTA, 0.5 mM PMSF, 0.5% Tween 20, 0.5% NonidentP40), and thensolubilized in 0.15 M KC 1 buffer C. Step 3: The enzyme was diluted to50 mM KC1 and loaded onto a pre-equilibrated HiTrap Heparin 5 mL columnat 1 mL per min flow rate. The column was washed with 10 volumes ofBuffer C (50 mM KC1), and protein eluted using a linear gradient 50 mMto 750 mnM KC1 (60 mL). Fractions (1 mL) were assayed for polymeraseactivity by measuring incorporation of [α³²P]dGTP at 70°C. usingactivated calf thymus DNA as a template with Mg²⁺ and all four dNTPsincluding [α³²P]dGTP. Peak fractions with WT and mutant enzymesconsistently eluted at approximately 300 mM KC 1, and were stored in 20%glycerol at-70° C.

[0080] Example 7. Kinetic Analysis of WT and Mutant Taq Pol I onIncorporating Ribonucleotide and Deoxyribonucleotide.

[0081] The efficiencies of purified WT, mutant #94, 265 and 346 Taq polI for incorporating dNTP and rNTP were determined by the followingprotocol. A 46mer template (5′-ccc ggg aaa ttt ccg gaa ttc cga tta ttgcta gcg gga att cgg cgc g, SEQ ID NO:37) was hybridized onto one of four³²P-labeled primers: 23 mer (5′-cgc gcc gaa ttc ccg cta gca at, SEQ IDNO: 35), 24mer (5′-cgc gcc gaa ttc ccg cta gca ata, SEQ ID NO: 38), 25mer (5′-cgc gcc gaa ttc ccg cta gca ata t, SEQ ID NO: 39) or 26 mer(5′-cgc gcc ccg gaa ttc ccg cta gca ata tc, SEQ ID NO: 40). Thesteady-state Michaelis-Menten parameters k_(cat) and K_(m) werecalculated by incubations with limiting amounts of Taq pol in thepresence of 5 nM primer/template and varying concentration of each dNTPor rNTP for 10 minutes at 55° C. as described in Boosalis, et al. (JBiol. Chem. 262:14689-14699 (1987)). All products were analyzed by 14%PAGE and quantified by phosphorimager analysis.

[0082] Kinetic analysis showed that purified WT Taq pol I did notefficiently incorporate ribonucleotides. WT Taq Pol I incorporated dG,dA and dC up to 30,000 times more efficiently (k_(cat)/K_(m)) than therespective ribonucleotides, and this difference was largely attributableto differences in K_(m) (Table 3). Taq pol I incorporatednoncomplementary nucleotides at a rate of 1 for each 9000 complementarydeoxynucleotides polymerized. (Tindall, et al., Biochemistry27:6008-6013 (1988)). Thus, Taq pol I is more efficient at excludingribonucleotides than excluding noncomplementary deoxynucleotides. Theactive site is especially adept at selecting dTTP over rUTP,incorporating dTTP 10⁶fold more efficiently relative to rUTP. These datasuggest DNA polymerases have evolved a sophisticated mechanism toexclude ribonucleotides, especially uracil, from its catalytic site. Incontrast, kinetic analysis of mutants (#94, 265 and 346) purified tohomogeneity showed that each polymerase incorporated rG, rA, and rC atan efficiency approaching up to {fraction (1/10)}th that of thecorresponding dNTP (Table 3). These mutants incorporated eachribonucleotide up to three orders of magnitude more efficiently than theWT polymerase. TABLE 3 Efficiency of dNTP and rNTP incorporation by WTand several mutant Taq pol I dNTP rNTP dNTP/rNTP Protein Nucleotidek_(cat) (s⁻¹) K_(m) (μM) k_(cat)/Km k_(cat) (s⁻¹) K_(m) (μM)k_(cat)/K_(m) Discrimination⁻* wild type G 0.020 0.021 1.0 0.0026 76 3.5× 10⁻⁵ 29,000 A 0.012 0.070 0.17 0.016 230 7.0 × 10⁻⁶ 24,000 C 0.0130.042 0.31 0.00083 59 1.4 × 10⁻⁵ 22,000 T/U 0.013 0.0050 2.6 0.00043 2401.8 × 10⁻⁶ 1,400,000 Mutant 94 G 0.0058 0.086 0.067 0.0065 0.94 0.007010 (A608S, I614N) A 0.012 0.15 0.080 0.0065 6.7 0.00097 83 C 0.00580.089 0.065 0.0075 14 0.00054 120 T/U 0.0071 0.022 0.32 0.0067 310.00022 1,500 Mutant 265 G 0.015 0.0071 2.1 0.012 0.77 0.016 130 (I614N,L616I) A 0.014 0.048 0.29 0.0080 4.4 0.0018 160 C 0.015 0.034 0.440.0073 6.2 0.0012 370 T/U 0.016 0.016 1.0 0.017 35 0.00049 2100 Mutant346 G 0.0020 0.12 0.017 0.0056 1.7 0.0032 5.3 (A608D, E615D) A 0.00400.20 0.020 0.0040 26 0.00015 130 C 0.0020 0.29 0.0069 0.0036 5.4 0.0006710 T/U 0.0087 0.018 0.48 — — — —

[0083] Example 8. Comparing the Efficiency of dGTP and rGTPIncorporation by WT and a Mutant.

[0084] WT Taq pol I (0.3 fmolμ/L for dNTP reactions and 3 fmol/μL forrNTP reactions) or mutant #94 (A6085, I614N; 0.2 fmol/μL for both dNTPand rNTP reactions) was incubated with 26mer/46mer (primer/template; 5nM) with increasing concentration of either dGTP or rGTP for 10 min at55 ° C. in 10 μL reactions. Product yield was quantified byphosphoimagery. The k_(cat)/K_(m) values obtained upon a hyperboliccurve fit of the plots reflects the efficiency of nucleotideincorporation. The results in FIG. 3 showed that incorporation of rGTPrelative to dGTP resulted in a product with a slower electrophoreticmigration.

[0085] Example 9. Determining the RNA Polymerase Activity of WT andMutants.

[0086] To determine if polymerases can fimction as RNA polymerases byincorporating multiple ribonucleotides sequentially, we incubatedpurified WT Taq pol I, mutant #265 (I614N and L616I), and mutant #346(A608D and E615D), in the presence of increasing amounts of all fourrNTPs (FIG. 4). While the WT enzyme inefficiently incorporated andextends ribonucleotides, both mutant enzymes polymerized multipleribonucleotides, even at rNTP concentrations well below that found incells. The strong pause sites produced at runs of template dAs wasexactly what one would predict from the kinetic data (Table 3),demonstrating decreased efficiency of UTP incorporation. Extension pastthese runs was facilitated by increasing incubation time or increasingribonucleotide concentrations. With Mn⁺² as the metal cofactor,elongation proceeded up to the 5′ end of the template even in presenceof low rNTP levels. In control incubations the elongated products weredegraded in alkali to regenerate the initial substrate, illustrating theproducts were RNAs.

[0087] Although the invention has been described with reference to thepresently preferred embodiments, it should be understood that variousmodifications can be made without departing from the spirit of theinvention.

1 40 1 7 PRT Thermus aquaticus VARIANT (7)...(7) X= at position 7 is avaline residue (Val) or an isoleucine residue (Ile) 1 Ser Gln Ile GluLeu Arg Xaa 1 5 2 8 PRT Thermus aquaticus 2 Asp Tyr Ser Gln Ile Glu LeuArg 1 5 3 13 PRT Thermus aquaticus 3 Leu Leu Val Ala Leu Asp Tyr Ser GlnIle Glu Leu Arg 1 5 10 4 7 PRT Esherichia coli 4 Tyr Ser Gln Ile Glu LeuArg 1 5 5 7 PRT Chlamydia trachomatis 5 Tyr Ser Gln Ile Glu Leu Arg 1 56 8 PRT Mycobacterium tuberculosis 6 Asp Tyr Ser Gln Ile Glu Met Arg 1 57 8 PRT Aquifex aeolicus 7 Asp Phe Ser Gln Ile Glu Leu Arg 1 5 8 8 PRTBorrelia burgdorferi 8 Asp Tyr Ser Gln Ile Glu Leu Ala 1 5 9 8 PRTRhodothermus obamensis 9 Asp Tyr Val Gln Ile Glu Leu Arg 1 5 10 8 PRTTreponema pallidum 10 Asp Tyr Thr Gln Ile Glu Leu Tyr 1 5 11 13 PRTEschericia coli 11 Leu Leu Val Ala Leu Asp Tyr Ser Gln Lys Glu Leu Arg 15 10 12 13 PRT Eschericia coli 12 Leu Leu Val Ala Leu Asp Tyr Ser GlnMet Glu Leu Arg 1 5 10 13 13 PRT Eschericia coli 13 Leu Leu Val Asp LeuAsp Tyr Ser Gln Met Glu Pro Arg 1 5 10 14 13 PRT Eschericia coli 14 LeuLeu Val Ser Leu Asp Tyr Ser Gln Asn Glu Leu Arg 1 5 10 15 13 PRTEschericia coli 15 Val Leu Val Ala Leu Asp Tyr Ser Gln Asn Glu Leu Arg 15 10 16 13 PRT Eschericia coli 16 Leu Leu Val Ala Leu Asp Tyr Ser LeuLys Glu Leu Arg 1 5 10 17 13 PRT Eschericia coli 17 Leu Leu Val Ala MetAsp Tyr Ser Gln Gln Glu Leu Arg 1 5 10 18 13 PRT Eschericia coli 18 LeuLeu Val Ala Leu Asp Tyr Ser Leu Gln Glu Leu Arg 1 5 10 19 13 PRTEschericia coli 19 Leu Val Val Ala Leu Asp Tyr Ser Gln Ile Asp Leu Arg 15 10 20 13 PRT Eschericia coli 20 Leu Leu Val Ala Leu Asp Tyr Ser ArgIle Glu Phe Arg 1 5 10 21 13 PRT Eschericia coli 21 Leu Leu Val Ala LeuAsp Tyr Ser Gln Asn Glu Ile Arg 1 5 10 22 13 PRT Eschericia coli 22 LeuLeu Val Gly Leu Asp Tyr Ser Gln Ile Asp Leu Arg 1 5 10 23 13 PRTEschericia coli 23 Leu Leu Val Val Leu Asp Tyr Ser Gln Ile Asp Leu Arg 15 10 24 13 PRT Escericia coli 24 Leu Leu Val Asp Leu Asp Tyr Ser Gln IleAsp Leu Arg 1 5 10 25 13 PRT Eschericia coli 25 Leu Leu Val Asp Val AspTyr Ser Gln Ile Asp Leu Arg 1 5 10 26 13 PRT Eschericia coli 26 Ile LeuLeu Ala Leu Asp Tyr Ser Gln Lys Glu Leu Arg 1 5 10 27 13 PRT Eschericiacoli 27 Leu Leu Met Ala Leu Asp Tyr Ser Gln Met Asp Leu Arg 1 5 10 28 13PRT Eschericia coli 28 Leu Leu Val Ala Leu Asp Phe Ser Gln Thr Asp LeuArg 1 5 10 29 13 PRT Eschericia coli 29 Leu Leu Val Val Val Asp Tyr SerGln Lys Glu Leu Arg 1 5 10 30 13 PRT Eschericia coli 30 Leu Leu Val AlaLeu Asp Tyr Ser Gln Thr Glu Phe Trp 1 5 10 31 13 PRT Eschericia coli 31Leu Leu Val Ala Leu Asp Phe Ser Gln Thr Asp Leu Arg 1 5 10 32 13 PRTEschericia coli 32 Leu Leu Met Val Val Asp Tyr Ser Gln Ile Asp Leu Arg 15 10 33 13 PRT Eschericia coli 33 Leu Leu Val Ala Leu Asp Tyr Arg GlnLys Asp Leu Met 1 5 10 34 13 PRT Eschericia coli 34 Arg Met Lys Ser IleAsp Tyr Arg Gln Ile Glu Leu Arg 1 5 10 35 23 DNA Eschericia coli 35cgcgccgaat tcccgctagc aat 23 36 43 DNA Eschericia coli 36 cggaagcttggctgcagaat attgctagcg ggaattcggc gcg 43 37 49 DNA Eschericia coli 37cccgggaaat ttccggaatt ccgattattg ctagcgggaa ttcggcgcg 49 38 24 DNAEschericia coli 38 cgcgccgaat tcccgctagc aata 24 39 25 DNA Eschericiacoli 39 cgcgccgaat tcccgctagc aatat 25 40 26 DNA Eschericia coli 40cgcgccgaat tcccgctagc aatatc 26

What is claimed is:
 1. A mutant DNA polymerase within the Pol I familyof polymerases, comprising a mutation in an active site of a naturallyoccurring DNA polymerase, wherein said active site comprises an aminoacid sequence LeuLeuValAlaLeuAspTyrSerGlnIleGluLeuArg (SEQ ID NO: 3),said mutation comprises an alteration of an amino acid other than Glu insaid sequence, and said mutant DNA polymerase possesses altered fidelityor altered catalytic activity in comparison with said naturallyoccurring DNA polymerase.
 2. The mutant DNA polymerase according toclaim 1, wherein said Asp of said amino acid sequence motif is notaltered in said mutant form.
 3. The mutant DNA polymerase according toclaim 1, wherein said mutant DNA polymerase incorporates aribonucleotide at a rate at least 10 fold greater than that of saidnaturally occurring DNA polymerase.
 4. The mutant DNA polymeraseaccording to claim 3, wherein said mutation comprises an alteration ofIle in said amino acid sequence.
 5. The mutant DNA polymerase accordingto claim 4, wherein said Ile is altered to a hydrophilic amino acid insaid mutant form.
 6. The mutant DNA polymerase according to claim 3,wherein said mutation comprises two or more amino acid substitution insaid amino acid sequence.
 7. The mutant DNA polymerase according toclaim 3, wherein said mutant DNA polymerase functions as both DNApolymerase and RNA polymerase.
 8. The mutant DNA polymerase according toclaim 1, wherein said polymerase incorporates an unconventionalnucleotide at a rate at least 10 fold greater than that of saidnaturally occurring DNA polymerase.
 9. The mutant DNA polymeraseaccording to claim 8, wherein said unconventional nucleotide is aribonucleotide analog.
 10. The mutant DNA polymerase according to claim8, wherein said unconventional nucleotide comprises a base labeled witha reporter molecule.
 11. The mutant DNA polymerase according to claim10, wherein said reporter molecule is a fluorophore or a hapten.
 12. Themutant DNA polymerase according to claim 8, wherein said unconventionalnucleotide is a chemotherapy drug.
 13. The mutant DNA polymeraseaccording to claim 12, wherein said chemotherapy drug is ara-C oracyclovir.
 14. The mutant DNA polymerase according to claim 8, whereinsaid unconventional nucleotide is an anti-viral or an anti-cancer drug.15. The mutant DNA polymerase according to claim 1, which has anincreased catalytic efficiency for incorporating deoxyribonucleotideover said naturally occurring DNA polymerase.
 16. The mutant DNApolymerase according to claim 1, wherein said naturally occurring DNApolymerase is a thermostable Thermus species DNA polymerase.
 17. Themutant DNA polymerase according to claim 16, wherein said Thermusspecies is Thermus aqaticus.
 18. The mutant DNA polymerase according toclaim 1, which possesses enhanced fidelity comparing with said naturallyoccurring DNA polymerase.
 19. An isolated nucleic acid sequence encodingthe mutant DNA polymerase according to claim
 1. 20. A mutant DNApolymerase within the Pol I family of polymerases, comprising a mutationin an active site of a naturally occurring DNA polymerase, wherein saidactive site comprises an amino acid sequenceLeuLeuValAlaLeuAspTyrSerGlnIleGluLeuArg (SEQ ID NO: 3), said mutationcomprises two or more amino acid substitutions, and said mutant DNApolymerase possesses altered fidelity or altered catalytic activity incomparison with said naturally occurring DNA polymerase.
 21. The mutantDNA polymerase according to claim 20, wherein said Asp of said aminoacid sequence motif is not altered in said mutant form.
 22. The mutantDNA polymerase according to claim 20, wherein said mutant DNA polymeraseincorporates a ribonucleotide at a rate at least 10 fold greater thanthat of said naturally occurring DNA polymerase.
 23. The mutant DNApolymerase according to claim 22, wherein said mutation comprises analteration of Ile in said amino acid sequence.
 24. The mutant DNApolymerase according to claim 23, wherein said Ile is altered to ahydrophilic amino acid in said mutant form.
 25. The mutant DNApolymerase according to claim 22, wherein said mutant DNA polymerasefunctions as both DNA polymerase and RNA polymerase.
 26. The mutant DNApolymerase according to claim 20, wherein said polymerase incorporatesan unconventional nucleotide at a rate at least 10 fold greater thanthat of said naturally occurring DNA polymerase.
 27. The mutant DNApolymerase according to claim 26, wherein said unconventional nucleotideis a ribonucleotide analog.
 28. The mutant DNA polymerase according toclaim 26, wherein said unconventional nucleotide comprises a baselabeled with a reporter molecule.
 29. The mutant DNA polymeraseaccording to claim 28, wherein said reporter molecule is a fluorophoreor a hapten.
 30. The mutant DNA polymerase according to claim 26,wherein said unconventional nucleotide is a chemotherapy drug.
 31. Themutant DNA polymerase according to claim 30, wherein said chemotherapydrug is ara-C or acyclovir.
 32. The mutant DNA polymerase according toclaim 26, wherein said unconventional nucleotide is an anti-viral drugor an anti-cancer drug.
 33. The mutant DNA polymerase according to claim20, which has an increased catalytic efficiency for incorporatingdeoxyribonucleotide over said naturally occurring DNA polymerase. 34.The mutant DNA polymerase according to claim 20, wherein said naturallyoccurring DNA polymerase is a thermostable Thermus species DNApolymerase.
 35. The mutant DNA polymerase according to claim 34, whereinsaid Thermus species is Thermus aqaticus.
 36. The mutant DNA polymeraseaccording to claim 20, which possesses enhanced fidelity comparing withsaid naturally occurring DNA polymerase.
 37. An isolated nucleic acidsequence encoding the mutant DNA polymerase according to claim 20.